For recombinant biopharmaceutical proteins to be acceptable for administration to human patients, it is important that residual impurities resulting from the manufacture and purification process are removed from the final biological product. These process components include culture medium proteins, immunoglobulin affinity ligands, viruses, endotoxin, DNA, and host cell proteins. These host cell impurities include process-specific host cell proteins (HCPs), which are process-related impurities/contaminants in the biologics derived from recombinant DNA technology. While HCPs are typically present in the final drug substance in small quantities (in parts-per-million or nanograms per milligram of the intended recombinant protein), it is recognized that HCPs are undesirable and their quantities should be minimized. For example, the U.S. Food and Drug Administration (FDA) requires that biopharmaceuticals intended for in vivo human use should be as free as possible of extraneous impurities, and requires tests for detection and quantitation of potential contaminants/impurities, such as HCPs. In addition, the International Conference on Harmonization (ICH) provides guidelines on test procedures and acceptance criteria for biotechnological/biological products. The guidelines suggest that for HCPs, a sensitive immunoassay capable of detecting a wide range of protein impurities be utilized. Although we and others have developed assays and reagents to detect immunoglobulins, DNA, endotoxins, viruses, and total HCPs, e.g., total Chinese hamster ovary proteins (CHOP) (reviewed in Chen A B, J Biotechnol in Healthcare 3:70-80 (1996); Krawitz et al., Proteomics 6:94-110 (2006)), there are currently no commercial reagents or analytical methods of sufficient specificity and sensitivity for the detection and quantification of single process-specific HCPs in recombinant protein preparations, such as immunoglobulin products, including those that co-purify with recombinant protein preparations.
In certain instances, significant dilution dependence may be observed when using immunoassays for the detection and quantification of total HCPs, e.g., total CHOP, suggesting such assays are not appropriate test procedures for accurate quantification of HCP impurities in a particular product. Investigation of such dilution dependence is important so as to enable the development of more appropriate test procedures. In certain instances, dilution dependence can be caused by “antigen excess” in which a single HCP species present in excess of the available antibodies accounts for the observed effects on assay performance (Anicetti et al., J. Immunol. Methods 91:213-224 (1986); Chen A B, J Biotechnol in Healthcare 3:70-80 (1996), Wang X, et al., Biotechnol Bioeng. 103(3):446-58 (2009)).
Sensitive analytical methods, such as LC-MS/MS can be used to identify and quantify single HCP species present in excess of available antibodies. Upon identification of such single HCP species, alternative assays of sufficient sensitivity and specificity and that are capable of being validated for approval by regulatory authorities and that can be used as a platform across multiple recombinant protein products, need to be developed.
In certain of our recombinant protein preparations produced in CHO cells, we identified an enzyme, phospholipase B-like 2, as a single CHOP species present in excess of available antibodies in a total CHOP ELISA assay. As used herein, “PLB2” and “PLBL2” and “PLBD2” are used interchangeably and refer to the enzyme “phospholipase B-like 2” or its synonym, “phospholipase B-domain-like 2”. Certain scientific publications on PLBL2 include Lakomek, K. et al., BMC Structural Biology 9:56 (2009); Deuschi, et al., FEBS Lett 580:5747-5752 (2006). PLBL2 is synthesized as a pre-pro-enzyme with parent MW of about 66,000. There is an initial leader sequence which is removed and potential 6 mannose-6-phosphate (M-6-P) groups are added during post-translational modification. M-6-P is a targeting modification that directs this enzyme to the lysosome via the M-6-P receptor. PLBL2 contains 6 cysteines, two of which have free sulfhydrals, and four form disulfide bonds. In acidic environments, PLBL2 is further clipped into the N- and C-terminal fragments having 32,000 and 45,000 MW, respectively. By analogy with other lysosomal enzymes, this cleavage is an activating step, allowing and access of the substrate to the active site.
There is about 80% PLBL2 amino acid sequence homology between hamster and human forms of the enzyme. The enzyme activity is thought to be to cleave either fatty acid chain from the phospholipids that make up cell membranes. There are other phospholipases with different substrate cleavage specificities. Similar enzymatic activities exist in microorganisms, where they are often a virulence factor. Although microorganisms have a similar enzymatic activity, the protein generating this activity is different, and there is low sequence homology between microbial and mammalian PLBL2 enzymes. Phospholipases produce free fatty acids (FFA) as one product of the substrate hydrolysis. Free fatty acids are themselves a potential immune-signaling factor. Dehydrogenation converts FFA to arachadonic acid which potentially participates in inflammation cascades involving eicosanoids.
Having identified PLBL2 as a single HCP (CHOP) in certain recombinant protein preparations, it would be highly advantageous and desirable to have reagents, methods, and kits for the specific, sensitive, and quantitative determination of PLBL2 levels in cell lines or in multiple products and at various stages of purification. Such reagents, methods and kits are particularly needed where there are no existing assays of sufficient consistency, sensitivity, specificity or efficiency. The invention described herein meets certain of the above-described needs and provides other benefits.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.